A continuous and expanding need exists for rapid, highly specific methods of detecting and quantifying chemical, biochemical, and biological substances. Of particular value are methods for measuring small quantities of pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, antibodies, metabolites, enzymes and nucleic acids.
The presence of these materials can often be determined by binding methods which exploit the high degree of specificity which characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques, and protein-ligand systems. In these methods, the existence of the complex of diagnostic value is typically indicated by the presence or absence of an observable label which has been attached to one or more of the complexing materials.
The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting a material of interest. A label is preferably inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without changing the important binding characteristics of those materials. Moreover, the label is preferably stable, and gives a highly characteristic signal. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are versatile, and can be detected at very low concentrations, However, they are expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Moreover, radioactive labels cannot be used in homogeneous methods. Disposal of radioactive waste is also of increasing concern both because of the potential risk to the public and the lack of radioactive waste disposal sites. The use of radioactive labeling is also time consuming, and can sometimes require as much as several days for detection of the radioactive label.
Enzyme labels and absorption-based detection instrument, e.g., ELISA, are safe, but lack sensitivity and stability for long term storage. Moreover, in enzyme immunoassay such as ELISA, a number of analysis steps are involved and a long period of time is required for the reaction. Fluorescent organic and inorganic molecules are safe and stable, but do not provide the same sensitivity as radio-isotope labels. With laser as an excitation source and complex optical detection, instrument cost is also a major disadvantage for fluorescent labels. Chemiluminescence and electrochemiluminescence provide high sensitivity for detection, but also employ optical detection and have relatively high instrument cost.
Photoelectrochemical labels for immunoassays have previously been described. For example, U.S. Pat. No. 4,293,310 describes an apparatus and method comprising a quencher and an electrochemical flow cell with a light means for determining the presence of photoelectrochemically labeled materials. Upon photoexcitation, the photoelectrochemically active label transfers an electron to a quencher molecule. The oxidized molecule is subsequently reduced with an electron from an electrode of the flow cell which is held at suitable potential. This electron is measured as photocurrent. The amount of free labeled analyte in the system is determined by the photocurrent signal. Although photoelectrochemical detection methods are cheaper than imaging devices employed in luminescence-based detection methods, this method has a limited detection range, and also suffers from interferents. (See Weber et al., Clin. Chem., 29:1665-1672 (1983)). Thus, there remains a need for analytical compositions and methods that are safe, stable, efficient, and inexpensive, and that provide a wide detection range.